This project focuses on continuing our X-ray crystallographic studies on Norovirus capsids and some of the non-structural proteins encoded by the norovirus genome that are critical for norovirus replication. Based on the substantial progress made in the previous funding period, the new specific aims in this proposal are designed to further our understanding of the structural basis of various virus related functions that could be helpful in the design of experimental strategies in Project 2, and identify novel targets for developing effective antiviral drugs for noroviruses in conjunction with proposed studies in Project 1. In Specific Aim 1, our focus will be to further investigate the structural basis of genotype-specific recognition of HBGA by noroviruses. In Specific Aim 2, our goal is to delineate the structural determinants of substrate specificity in the NV protease, making use of a novel system that we discovered during the current grant period. In addition, we will perform structural analysis on small molecule inhibitors identified in Project 1 in complex with the protease. These structures will facilitate the design of inhibitors with increased potency. In Specific Aim 3, we will determine the structure of a norovirus VPg. Several studies on both animal andtlhuman caliciviruses have strongly implicated this protein as a cap substitute for translation initiation of the viral RNA and in a primer function during genome replication. We have expressed and purified VPg, VPg-Pro, VPg-Pro-Pol and obtained crystals of VPg-Pro demonstrating the feasibility of obtaining the structure of this important protein and setting the stage for structural studies to understand the molecular basis of its interaction with ribosomal initiation factors and viral polymerase, and the mechanism of uridylylation. In Specific aim 4, we will determine the structure ofthe p41 of Norwalk virus. p41 protein is an NTPase/helicase analogous to picornavirus 2C protein. Based on sequence analysis, p41 exhibits motifs such as A, B, B', C and Arg finger found in SF3-2 helicases. Although, the function of this protein is unclear, one hypothesis is that it provides a mechanism to prevent viral ssRNA from annealing during the strand synthesis by the viral polymerase. We have been able to express and purify this protein to homogeneity.